An In Vitro Assay for Monitoring the Formation and Branch Migration of Holliday Junctions Mediated by a Eukaryotic Recombinase

Abstract

DNA strand exchange is a core reaction of homologous recombination directly catalyzed by Rad51/Dmc1 RecA family recombinases in eukaryotes. This reaction proceeds through the formation of several DNA intermediates. The X-shaped four-way DNA structure known as a Holliday junction (HJ) is a central intermediate in homologous recombination. Genetic and biochemical studies indicate that the HJ is important for the production of crossover-type recombinants, which are reciprocal exchange products. According to a recombination model for the repair of DNA double-strand breaks, the formation of HJs requires a reciprocal duplex–duplex DNA exchange known as the DNA four-strand exchange reaction. In vitro analyses using purified recombination proteins and model DNA substrates provide a mechanistic insight into the DNA strand exchange reaction, including the steps leading to the formation and branch migration of Holliday junctions.

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Acknowledgments

We thank K. Ito and T. Koizumi for preparation of the manuscript. This study was supported in part by grants in aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MECSST) of Japan and for scientific research (A) to HI and for young scientist (B) to YM from the Japan Society for the Promotion of Science (JSPS), and by the Uehara Memorial Foundation to HI.